1. Field of the Invention
The present invention relates to a system comprising a modified bacterial gene for cytosine deaminase that has been engineered into a eukaryotic expression vector and the expression of the gene by mammalian cells.
The present invention further relates to methods, gene therapies and vaccines that employ the negative selectable marker, cytosine deaminase, which has the ability to produce a toxic antimetabolite 5-fluorouracil from 5-fluorocytosine.
2. Background Information
Selectable genetic markers are important tools in the study of the regulation and function of genes and are potentially important in gene transfer therapies. Conferring unique resistance or sensitivity to cytotoxic agents enables the skilled artisan the ability to select or delete genetically altered cells from a mixed population.
The enzyme cytosine deaminase (CD) is useful in the present invention as a selectable genetic marker because of its ability to catalyze the deamination of cytosine to uracil (M. Kilstrup et al., J. Bacteriology 171:2127-2127 (1989); L. Anderson et al., Arch. Microbiol. 152:115-118 (1989)). Bacteria and fungi which express this gene convert 5-fluorocytosine (5FC) to 5-fluorouracil (5FU) and this metabolite is toxic to the microorganism (A. Polak and H. J. Scholer, Chemotherapy (Basel) 21:113-130 (1975)). Mammalian cells do not express significant amounts of cytosine deaminase and do not deaminate 5FC (A. Polak et al., Chemotherapy 22:137-153 (1976); B. A. Koechlin et al., Biochemical Pharmacology 15:434-446 (1966)); 5FC is relatively nontoxic to them (J. E. Bennett, in Goodman and Gilman: the Pharmacological Basis of Therapeutics. 8th ed., eds. A. G. Gilman, T. Rall, A. S. Nies and P. Taylor (Pergamon Press, New York) pp. 1165-1181). However, 5FU has potent cytotoxic effects on mammalian cells. 5FU is subsequently metabolized to FUTP and FdUMP and thereby inhibits both RNA and DNA synthesis and kills the cell (P. Calabrisi and B. A. Chabner in Goodman and Gilman: the Pharmacological Basis of Therapeutics. 8th ed., eds. A. G. Gilman, T. Rall, A. S. Nies and P. Taylor (Pergamin Press, New York) pp. 1209-1263); L. E. Damon et al., Pharmac. Ther. 43:155-189 (1989)). Thus, intracellular metabolic conversion of 5FC to 5FU should be lethal to mammalian cells.
The bacterial gene for cytosine deaminase has recently been isolated and cloned (L. Anderson et al., (1989)). The present invention provides a new negative selectable marker in which the gene for cytosine deaminase from a microorganism, of which bacteria is an example, is modified and integrated into a eukaryotic expression vector and expressed in mammalian cells conferring upon the transfected cells a unique susceptibility to the cytotoxic effects of 5FC. The present invention also provides methods that use the cytosine deaminase negative selection system in vitro to selectively eliminate subpopulations of cells, and in vivo for gene transfer therapies and vaccines.